Dr. Cushion has recently succeeded in establishing an axenic culture system which supports limited growth of Pc, similar to that achieved with tissue cultures. It is the overall goal of this project to axenically culture Pc. The first specific aim is to attempt to identify isolation procedures that produce the healthiest organisms for culture. To expedite culture development, the investigator also plans to develop rapid methods for evaluation of organism numbers and viability. These methods will then be applied to accomplish the second goal, to axenically culture Pc. The investigator will attempt to develop a medium which supports the growth of Pc by following nutritional evaluations and biochemical analyses used by other investigators who have successfully established such cultures for other protists. Various concentrations of blocks of nutrients commonly required for culture of microbes will be evaluated for Pc growth in the present axenic medium or an improved crude medium. Optimal concentrations of each block will be identified by narrowing the range of dilutions. Once identified, the optimal concentration of a block will remain constant while another block is tested and optimized. Growth and viability will be assessed by a variety of methods including radiolabeled precursor incorporation, enumeration of organisms and fluorometric assay. The third goal of the project is to try to characterize the life cycle stages and kinetics of growth in axenic culture. Ultrastructural and kinetic analyses will be used to identify the life cycle stages replicating in cultures, determine the growth rate, and monitor the health of the organisms. Since parasitic protists commonly decrease their virulence when cultured over long periods and this virulence has oftentimes been linked to surface antigens, the antigenicity of the cultured organisms will be ultrastructurally evaluated. The projects have been designed to provide, first, new or improved methods of assessing organism number and viability. Secondly, it is anticipated that information on the physiologic state of organisms isolated from infected lungs will be obtained. Lastly, it is hoped that increased understanding of the metabolic capabilities of Pc and, in addition, the development of axenic culture for Pc, will be obtained.